Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.


NACT
NACT + P Distance from CK + Cas3a -to Distance from CK + Cas3a + to   (right, N=31 patients).The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Statistical significance was evaluated using two-sided Wilcoxon signed-rank tests.Unadjusted p-values are showed on the plot for CK+Ki67+ unadjP=4.53e-06 in NACT unadjP=9.09e-13 in NACT+P.C. Box and dotplots representing, for each patient, the ratio between the post-treatment and the pre-treatment nearest neighbor distance (µm) from CK+Cas3a-or CK+Cas3a+ cells to CD4+, CD8+, CD4+PD-1+, CD8+PD-1+, CD4+PD-1+Ki67+, and CD8+PD-1+Ki67+ cells in patients receiving NACT alone (blue), N=21 patients, and patients receiving NACT+P (red), N=31 patients.The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Statistical significance was evaluated using two-sided Wilcoxon rank sum test.Distance from CK+Cas3a+ to CD8+PD-1+, unadjP=6.25e-03.D. Neoantigen-specific tumor infiltrating lymphocyte scores25 showing tumors with (N=22, blue) or without (N=34, yellow) an increase in PD-1+ CD8+ lymphocytes after treatment.The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Statistical significance was evaluated using two-sided Wilcoxon rank sum test and unadjP=1.78e-03is displayed.E. Box and dot plots representing the number of cells/mm2 (i.e., the density) of CD4+PD-1+Ki67+ and CD8+PD-1+Ki67+ cells both in the tumor and stroma pre-and post-treatment in the NACT arm (left, N=21 patients) and the NACT+P arm (right, N=31 patients).The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Statistical significance was evaluated using two-sided Wilcoxon signed-rank test.Source data are provided as a Source Data file.

Predictive biomarkers of response
to NACT + Pembrolizumab. A. Representative image of the multiplex IF staining focusing on B cells, tertiary lymphoid structures and antibody-secreting cells.B.C.D. Boxplots representing the scaled expression scores of various transcriptomic signatures from MCP-counter and quanTIseq (B), the densities of various immune cell subsets in the tumor versus stroma (C) and the % of tumor surface occupied by TLS (D), in non progressors, NPr, (green-blue, N=7 NACT and N=13 NACT+P) versus progressors, Pr, (dark yellow, N=13 NACT and N=19 NACT+P) from NACT arm (top) vs NACT+P arm (bottom).The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Significant differences using the two-sided Wilcoxon rank sum test are highlighted with stars on the top (unadjP).Unadjusted p-values for NACT+P displayed on B, QuanTIseq Macrophages M2=0.037;Quantiseq Monocytes=0.006;CD8B/FOXP3=0.008;MCP Neutrophils=0.045;MCP Endothelial cells=0.027.Source data are provided as a Source Data file.
Gating strategy for determination of CD39 expression on T cell populations in human ovarian cancer.After the exclusion of debris, doublets cells (by FSC/SSC plots) and dead cells (LDAqua + ), immune cells were selected on CD45 expression (CD45 + ).The different populations of T cells were gated by the expression of CD8 and CD4 and Teff and Treg were distinguished based on FOXP3 expression (FOXP3 neg = Teff and FOXP3 + = Treg).Then CD39 expression was analyzed on each population.Percentage refer to the proportion of cells in the previous parent gate.

FOXP3 gene expression ratio is associated with response to NACT+P. A.
Spearman correlation between density of intra-epithelial CD8+ T cells and CD8B/FOXP3 gene expression ratio.Error bands represents the 95% CI as a shaded gray area.B.F.G.PFS (left) and OS (right) curves according to the density of intra-epithelial (ie) CD8+ T cells before treatment (B), the CD8B/TIGIT gene expression ratio (High ** ** Le Saux et al.Supplementary Figure 6

High expression of KDR/VEGFR2 is associated with resistance to NACT+P
. A.PFS (left)and OS (right) curves of patients that received Bevacizumab included in the NeoPembrOv trial according to the expression of the endothelial gene signature (High NACT (red), n=11; Low NACT (dark blue), n=9; High NACT+P (orange), n=19; Low NACT+P (light blue), n=10).Patients were stratified based on the best cutoff.Statistical comparison of survival curves for NACT+P Low vs. NACT Low was performed using the likelihood ratio test.B. Association between the expression in TPM of genes included in the MCP endothelial cell signature and patient status (progressors (Pr) in dark yellow vs non progressors (NPr) in green-blue).The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Significant differences using the two-sided Wilcoxon rank sum test are highlighted with stars on the top (unadjP).Association between the expression in TPM of genes encoding for VEGFR2/KDR ligands and patient status (progressors (Pr) in dark yellow vs non progressors (NPr) in green-blue).The centerline of boxes depicts the median values; the bottom and top box edges correspond to the first and third quartiles.Significant differences using the two-sided Wilcoxon rank sum test are highlighted with stars on the top (unadjP).E. PFS (left) and OS (right) curves according to PDGFA gene expression (High NACT (red), n=11; Low NACT (dark blue), n=10; High NACT+P (orange), n=22; Low NACT+P (light blue), n=10).Patients were stratified based on the best cutoff.Statistical comparison of survival curves for NACT+P Low vs. NACT Low was performed using the likelihood ratio test.Source data are provided as a Source Data file.

Association of selected gene expression ratios with response to the combination of NACT+P.
TIL10 signature matrix showing the top 5 genes with the higher expression in monocytes and positive log2 fold change compared to M2-like macrophages.Le Saux et al.Supplementary Table3Gene symbols italicized Two-sided Wilcoxon rank sum test unadjusted p-values without correction for multiple testing.